Empagliflozin–pirfenidone dual therapy improves cardiac function and structure in a preclinical two-hit HFpEF model

Figures

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  FIGURE 1. Induction of HFpEF in rats using a high-fat diet and L-NAME (A) Representative left ventricular M-mode echocardiographic images illustrating cardiac structure and function in control and HFpEF-induced rats following 5 weeks of L-NAME and high-fat diet administration. (B) Quantitative analysis of systolic function parameters, including left ventricular ejection fraction (LVEF), stroke volume (SV), and cardiac output (CO), demonstrating significant impairment in HFpEF rats (p < 0.005 versus Chow). (C) Increased left ventricle posterior wall diameter (LVPWd) observed in HFpEF rats. (D) Reduced fractional shortening (FS), predominantly in male rats subjected to HFD + L-NAME, indicating a sex-related difference (p < 0.003 versus control within male rats). (E,F) Diastolic function assessment via mitral Doppler echocardiography showing a significantly decreased E/A ratio, along with prolonged isovolumetric relaxation time (IVRT) and deceleration time (DT) in HFpEF rats (p < 0.005 versus chow). (G) Exercise tolerance, evaluated by treadmill running distance, is markedly reduced in HFpEF rats (p < 0.0001 versus Chow). Data are expressed as mean ± SEM, pooled from both sexes in the absence…

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  FIGURE 2. Structural and histological changes in HFpEF rats. (A) Representative Masson’s trichrome staining of mid-ventricular short-axis sections (scale bar = 1 mm) and quantification of left ventricular (LV) mass demonstrate increased myocardial fibrosis and LV mass in HFpEF rats (p = 0.0135 vs. Chow). (B) The left ventricle weight index (LVWI) and lung wet-to-dry weight ratio (LWDR) are also elevated. (C) Representative images of myocyte cross-sectional area (CSA) confirm cardiomyocyte hypertrophy following HFD + L-NAME administration (p < 0.0001 vs. chow). (D) Collagen volume fraction (CVF) analysis indicates increased fibrotic deposition in the subendocardium (p < 0.001 vs. chow), myocardium (p = 0.003 vs. chow), and subepicardium (p < 0.001 vs. chow) in HFpEF rats. All histological images are derived from heart sections of male rats. Data are presented as mean ± SEM, pooled from both sexes when no interaction between sex and L-NAME was observed, as determined using two-way ANOVA with Tukey’s post hoc test. Significance was set at p < 0.05.

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  FIGURE 3. Impact of empagliflozin and pirfenidone on HFpEF outcomes (A) Study design schematic outlining HFpEF induction, treatment groups, and endpoints. (B,C) Representative echocardiographic M-mode images and quantification of LVEF, CO, and SV reveal significant improvements in EMPA + PFD-treated rats (p < 0.05 vs. untreated HFpEF), demonstrating improved cardiac systolic function in EMPA + PFD-treated rats compared to untreated HFpEF controls. (D) The benefit of EMPA + PFD in fractional shortening was only observed in male rats (P = 0.05 vs. untreated HFpEF). (E,F) Diastolic function indices, including E/A ratio, IVRT, and DT, are significantly improved following EMPA + PFD treatment (p < 0.05 vs. untreated HFpEF). (G) Exercise tolerance test results show a significant increase in running distance in EMPA + PFD-treated rats (p < 0.0001 vs. untreated HFpEF rats). Representative echocardiographic images presented in the figure were taken from male rats. Data are presented as mean ± SEM of the pooled dataset of both sexes; differences were analyzed using two-way ANOVA with Tukey’s post hoc test, with significance determined at p < 0.05. Iconography in this figure was sourced from…

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  FIGURE 4. Histological evidence of reduced hypertrophy and fibrosis with EMPA + PFD treatment. (A) Masson’s trichrome staining shows reduced myocardial fibrosis, and left ventricular mass is significantly lower in EMPA + PFD-treated rats compared to HFpEF controls (p < 0.05). (B) When compared to control, left ventricle weight index (LVWI) was significantly lower in EMPA + PFD (adjusted p < 0.001), while difference in the lung wet-to-dry weight (LWDR) ratio was modest (adjusted p < 0.05). (C) Myocyte cross-sectional area (CSA) measured using WGA indicates reduced hypertrophy in EMPA + PFD-treated rats compared to HFpEF controls (p < 0.001). (D) Collagen volume fraction (CVF) analysis shows that EMPA + PFD treatment significantly reduces fibrosis, particularly in the mid-wall (p = 0.0267 vs. untreated HFpEF) and endocardium (p = 0.0021 vs. untreated HFpEF), with no significant changes in the subepicardial region. All histology images are presented using heart sections from male rats. Data are expressed as mean ± SEM, with two-way and Tukey’s post hoc tests used for analysis.

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  FIGURE 5. RNA sequencing reveals distinct gene expression profiles in EMPA + PFD-treated HFpEF male rats. (A) Differential expression analysis comparisons (adjusted p-value < 0.05 and absolute fold change >2). Bar chart presenting the number of upregulated and downregulated DEGs for EMPA + PFD, PFD, EMPA, and control (9w) vs. untreated (left) or vs. HFpEF (middle), highlighting significant transcriptional shifts. (B) Principal component analysis (PCA) demonstrating clustering of gene expression profiles, with EMPA + PFD-treated rats showing a shift toward the healthy control (5w) phenotype. (C) Venn diagram illustrating the overlap of DEGs between disease establishment (DE), physiological changes (PC), untreated (UT), and treatment effect (TE) comparisons. (D) Heatmap of hierarchical clustering of DEGs across study groups, illustrating upregulation and downregulation patterns. (E) K-means clustering analysis of enriched Gene Ontology (GO) biological processes, with pathways associated with protein kinase C-activating G-protein-coupled receptor, inflammation, and metabolism showing significant modulation in EMPA + PFD-treated rats. Data were analyzed using DESeq2, with an FDR cut-off of…

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  FIGURE 6. PKC–GPCR signaling pathway dysregulation drives NF-κB-mediated myocardial inflammation in HFpEF and is attenuated by EMPA + PFD. (A) Overview of DEGs of PC, DE, UT, and TE specific to the PKC–GPCR signaling pathway. Bubble plot displaying Log2FC with corresponding adjusted p-values (Padj) for each gene across four experimental comparisons: physiological cardiac maturation (PC: healthy 9-week vs. healthy 5-week rats), disease establishment (DE: HFpEF vs. healthy rats at 5 weeks), untreated chronic disease (UT: HFpEF vs. healthy rats at 9 weeks), and treatment effect (TE: treated vs. untreated HFpEF rats at 9 weeks). (B) Western blot analysis of phospho-PKC (Pan/βII-Ser660) and phospho-NF-κB p65 (Ser536) with GAPDH as a loading control. Semiquantitative analysis of the respective proteins was normalized to GAPDH expression (n = 3). Differences were analyzed using one-way ANOVA followed by Tukey’s post-hoc test. (C) Immunofluorescence staining of heart sections demonstrating nuclear translocation of NF-κB in cardiac troponin T-positive cardiomyocytes. Statistical significance was determined using ANOVA with post-hoc testing, with p < 0.05 considered significant.